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The stimulation by phytohemagglutinin (PHA) of DNA synthesis in cultured blood lymphocytes of guinea pig was markedly inhibited by addition of leupeptin, a well-characterized, powerful protease inhibitor of tripeptide nature. About 30 to 40 per cent inhibition was observed at 40 μg/ml of leupeptin when leupeptin was added 30 min prior to or together with PHA. Per cent inhibition by the appropriate amount of leupeptin was proportional to the amount of PHA added in the range of 0.6 to 3.0 μg of PHA at which the per cent inhibition reached maximum. This inhibitory effect of leupeptin on PHA stimulation was abolished when the lymphocytes were preincubated with PHA for more than 10 min before addition of leupeptin or preincubated with leupeptin for more than 60 min prior to PHA addition.

VARIOUS substances have been reported to activate sea urchin spermatozoa, and to increase either their respiration or motility. It has been reported that sea urchin spermatozoa treated with jelly coat solution, that is, the so-called fertilizin, show an outburst of motility1 and an increase of oxygen consumption2. Hathaway obtained an egg substance from Arbacia eggs free of jelly which increased sperm respiration and motility to a great extent. This substance was reported to be diffusible on dialysis, soluble in alcohol, heat stable and non-volatile3. Mohri4, however, reported that 0.5 per cent soy bean lecithin or egg lecithin increased the oxygen uptake when these substances were added after 90 min of incubation. He also found a great increase of respiration by octanoate but little effect by glucose and fructose, and it was suggested that, unlike mammalian spermatozoa which preferentially utilize carbohydrates as energy source, the activity of sea urchin spermatozoa depends on a non-glycolytic, presumably lipid metabolism, because a considerable decrease of endogenous phospholipids was also observed during ageing4.

A COLOUR reaction, known as Scherer's test, is extensively used as a specific, qualitative test for inositol. It is based on the formation of the coloured alkaline earth metal salt (usually calcium salt) of rhodizonic acid on the oxidation of inositol with concentrated nitric acid1,2. While studying phos-pholipids containing inositol we needed to identify accurately very small amounts of inositol by paper chromatography, or by spot tests on filter paper. P. Fleury et al. 3 reported a method in which they used mercuric oxide as a catalyst of the oxidation, and barium acetate as for the alkaline earth metal. This method, however, is tedious and sometimes gives uncertain results; the smallest quantity detectable is 5–10 µgm. This may be ascribed to the formation of yellow mercuric salt at the first stage of the reaction. A more sensitive, reliable and easier method was, therefore, devised by modifying that of Fleury. This modified method can, in addition, be applied to the detection of inositol diphosphate located on paper chromatograms. The procedure is as follows.